1. Field of the Invention
The present invention relates to a novel gene encoding a family of proteins, called Smad, which intracellularly transduce stimuli elicited by a physiologically active substance belonging to the transforming growth factor beta (hereinafter referred to as TGF.beta.) family.
2. Description of the Prior Art
The TGF.beta. family is a group of peptidic physiologically active substances widely distributed in the animal kingdom. This family include very important physiologically active peptides such as TGF.beta. found as a substance involved in proliferation of tumor cells, bone morphogenetic protein (BMP) that has a significant function for bone formation in vertebrates including human, inhibin that regulates the secretion of follicle-stimulating hormone from pituitary gland, activin that possesses activity as an erythroide differentiation factor, and neurotrophic factor derived from glial cells (Bock, G. R. and Marsh, J. eds., 1991, Clinical Application TGF.beta., Ciba Foundation Symposium, Johns Wiler & Sons). A clue to the solution of the question of how the TGF.beta. family peptides act on cells was initially obtained by cDNA cloning of receptors for these peptides and by determining their nucleotide sequences together with the amino acid sequences deduced therefrom. Receptors for this family have a transmembrane-type protein-phosphotransferase activity (protein kinase activity) specific to serine (and threonine) residues. This fact demonstrates that phosphorylation of intracellular protein is involved in transduction of stimuli from the TGF.beta. family (Sporn, M. B. and Roberts, A. B. eds., 1990, "Peptide Growth Factors and Their Receptors", part I and II, Springer-Verlag, Berlin).
As factors mediating stimuli from the TGF.beta. family, genes designated as Mad and Sma are hitherto known in Drosophila and Nematoda, respectively. In recent years, several genes showing homology to Mad and Sma have been found in several vertebrates including human. Their cDNAs have been cloned and their nucleotide sequences were determined. Those genes and the proteins encoded by the same were named Smad, and to date, Smad1, Smad2, Smad3, Smad4, Smad5, and Smad6 have been reported (Derynck R. et al., 1996, "Nomenclature: Vertebrate mediators of TGF.beta. family signals", Cell, 18, 173). Furthermore, during early embryogenesis, Smad1 is known essential for a basic and significant determination as for which side of the embryo becomes dorsal and the other becomes ventral (Graff, J. M. et al., 1996, Cell, 85, 479-487). It has been shown that inactivation of Smad2 gene is one of the causes for colorectal cancer in human, while Smad4 gene is shown to be identical to a tumor suppressor gene DPC4 that is known to be strongly associated with repression of pancreatic cancer (Eppert, K., et al., 1996, Cell, 86, 543-552; Hahn, S. A. et al., 1996, Science, 271, 350-353). Thus, the Smad protein family may be signal transduction factors which transduce stimuli from physiologically active peptides of the TGF.beta. family, while they may also be factors profoundly involved in generation of cancer.
The known Smad family proteins are intracellular proteins consisting of about 400-550 amino acid residues which have an amino- and a carboxy-terminal regions relatively well conserved among the family. As a consequence of increased kinase activity of the specific receptors induced by the TGF .beta. family stimulus, Smad proteins are rapidly phosphorylated and concentrated into nucleus. In the nucleus, the area of gene transcription, Smad proteins uniquely regulate gene expression through unknown mechanism mediated by oligomer formation among the same or different kinds of molecules (Massagu J., 1996, Cell, 85, 947-950).
In recent years, it has been found out that a variety of physiologically active substances like TGF.beta., including hormones and cytokines, function eventually through regulation of gene expression in the target cells. Specificity of activity of each physiologically active substance is determined by the nature of receptor and subsequent signal transduction factor for the particular substance. In addition, a signal caused by a single physiologically active substance often activate several kinds of signal transduction factors, which results in branching of the transduction pathway. Isolation of signal transduction factors and elucidation of their properties are therefore helpful to understand mechanisms through which various physiologically active substances function, and to employ the factors as targets for pharmaceuticals.
As described above, the TGF.beta. family members play very important roles in various physiological events including growth control, immune response, cell differentiation, morphogenesis during embryo and the like. More than 50 physiologically active substances belonging to the TGF.beta. family are hitherto known, and they include substances of which deficiency or excess in quantity, or abnormality in quality is known to be associated with pathologies related to the above physiological events, such as cancer, autoimmune disease, osteoporosis, anemia, congenital deformity and the like. Similarly, genetic analyses have also shown that defects in the Smad family, which transduces stimuli (signals) from the TGF.beta. family, is involved in various abnormalities or pathologies, for example, in cancer which is the highest cause of death in advanced countries including many of Europe and North America. For prevention or treatment of cancer, it is desirable to elucidate all of the genes associated with cancer. However, relatively small number of Smads have been hitherto identified when compared with the already known physiologically active substances belonging to the TGF.beta. family. This fact suggests that there remain unidentified Smad family members. Therefore, isolation of a novel Smad gene will enable us to find a further pathway involving the TGF.beta. family, and such a gene is expected to be useful as a diagnostic agent for detecting abnormalities, such as tumor, at gene level.
The present invention aims to provide a novel factor belonging to the Smad family which transduces a signal of a physiologically active substance of the TGF.beta. family, and to provide a gene encoding said factor.
In view of the important role, in biological responses, of TGF.beta. peptides and their signal transducer Smad family proteins, the present inventors screened cDNAs derived from Mus musculus in order to clone a novel Smad gene. As a result, cDNA clones which correspond to mRNA encoding a novel Smad family protein were identified in a cDNA mixture derived from the whole tissue of the 17-day embryos. The present invention has been completed on the basis of this finding.